Chromatin structure plays an important role in the modulation of gene expression in eukaryotic development. During mammalian spermiogenesis the histones packaging the haploid male genome are modified and eventually replaced by protamines which are arginine rich proteins with a high content of cysteinyl residues. The mammalian sperm genome is transcriptionally inert, unlike the corresponding genome of the egg. However, the sperm nucleus, once it enters the oocyte at fertilization is rapidly processed by unknown mechanisms and paternally coded proteins may be detected by the two cell embryo. The processing reactions appears to involve reduction o the protamine disulfide bonds, decondensation of the tightly packaged sperm chromatin and reconstitution of the paternal genome into nucleosome structures. The present studies have used Staphyloccocal nuclease to probe the organization of the mammalian sperm nucleus and have demonstrated that isolated nuclei are resistant to cleavage. However, following reduction and alkylation, 30% of the sperm DNA could be digested and the remaining DNA had a heterodisperse size distribution. By morphological criteria, a model acidic protein, polyglutamic acid was capable of decondensing purified sperm nuclei that had been reduced and alkylated and the maximal extent of nuclease digestion increased to 85-90%. The subsequent addition of purified, exogenous core histones in 0.1 M NaC1 partially reversed this vulnerability to nuclease cleavage such that only 55% of the DNA was digested. Furthermore, analysis of the remaining DNA revealed a nucleosome ladder pattern with unit length repeat of 150 base pairs. These results strongly suggest that polyglutamic acid can mediate not only decondensation of sperm nuclei but also the assembly of sperm chromatin into nucleosomes. This system will be used as an assay for the macromolecules that mediate these reactions in vivo.